Browsing by Author "Bronfman, Francisca C."

Now showing 1 - 11 of 11
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    An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons
    (2020) Stuardo, Nicolas; Moya-Alvarado, Guillermo; Ramirez, Carolina; Schiavo, Giampietro; Bronfman, Francisca C.
    Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
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    BDNF/TrkB signaling endosomes in axons coordinate CREB/mTOR activation and protein synthesis in the cell body to induce dendritic growth in cortical neurons
    (2023) Moya-Alvarado, Guillermo; Tiburcio-Felix, Reynaldo; Ibanez, Maria Raquel; Aguirre-Soto, Alejandro A.; Guerra, Miguel, V; Wu, Chengbiao; Mobley, William C.; Perlson, Eran; Bronfman, Francisca C.
    Brain-derived neurotrophic factor (BDNF) and its receptors tropomyosin kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the CNS. After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation.
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    c-Abl Tyrosine Kinase Is Required for BDNF-Induced Dendritic Branching and Growth
    (2023) Chandia-Cristi, America; Stuardo, Nicolas; Trejos, Cristian; Leal, Nancy; Urrutia, Daniela; Bronfman, Francisca C.; Rojas, Alejandra Alvarez
    Brain-derived neurotrophic factor (BDNF) induces activation of the TrkB receptor and several downstream pathways (MAPK, PI3K, PLC-gamma), leading to neuronal survival, growth, and plasticity. It has been well established that TrkB signaling regulation is required for neurite formation and dendritic arborization, but the specific mechanism is not fully understood. The non-receptor tyrosine kinase c-Abl is a possible candidate regulator of this process, as it has been implicated in tyrosine kinase receptors' signaling and trafficking, as well as regulation of neuronal morphogenesis. To assess the role of c-Abl in BDNF-induced dendritic arborization, wild-type and c-Abl-KO neurons were stimulated with BDNF, and diverse strategies were employed to probe the function of c-Abl, including the use of pharmacological inhibitors, an allosteric c-Abl activator, and shRNA to downregulates c-Abl expression. Surprisingly, BDNF promoted c-Abl activation and interaction with TrkB receptors. Furthermore, pharmacological c-Abl inhibition and genetic ablation abolished BDNF-induced dendritic arborization and increased the availability of TrkB in the cell membrane. Interestingly, inhibition or genetic ablation of c-Abl had no effect on the classic TrkB downstream pathways. Together, our results suggest that BDNF/TrkB-dependent c-Abl activation is a novel and essential mechanism in TrkB signaling.
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    Characterization of an Agarophyton chilense Oleoresin Containing PPARγ Natural Ligands with Insulin-Sensitizing Effects in a C57Bl/6J Mouse Model of Diet-Induced Obesity and Antioxidant Activity in Caenorhabditis elegans
    (2021) Pinto, Claudio; Raquel Ibanez, Maria; Loyola, Gloria; Leon, Luisa; Salvatore, Yasmin; Gonzalez, Carla; Barraza, Victor; Castaneda, Francisco; Aldunate, Rebeca; Contreras-Porcia, Loretto; Fuenzalida, Karen; Bronfman, Francisca C.
    The biomedical potential of the edible red seaweed Agarophyton chilense (formerly Gracilaria chilensis) has not been explored. Red seaweeds are enriched in polyunsaturated fatty acids and eicosanoids, which are known natural ligands of the PPAR gamma nuclear receptor. PPAR gamma is the molecular target of thiazolidinediones (TZDs), drugs used as insulin sensitizers to treat type 2 diabetes mellitus. Medical use of TZDs is limited due to undesired side effects, a problem that has triggered the search for selective PPAR gamma modulators (SPPARMs) without the TZD side effects. We produced Agarophyton chilense oleoresin (Gracilex(R)), which induces PPAR gamma activation without inducing adipocyte differentiation, similar to SPPARMs. In a diet-induced obesity model of male mice, we showed that treatment with Gracilex(R) improves insulin sensitivity by normalizing altered glucose and insulin parameters. Gracilex(R) is enriched in palmitic acid, arachidonic acid, oleic acid, and lipophilic antioxidants such as tocopherols and beta-carotene. Accordingly, Gracilex(R) possesses antioxidant activity in vitro and increased antioxidant capacity in vivo in Caenorhabditis elegans. These findings support the idea that Gracilex(R) represents a good source of natural PPAR gamma ligands and antioxidants with the potential to mitigate metabolic disorders. Thus, its nutraceutical value in humans warrants further investigation.
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    Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann-Pick type C disease
    (WILEY, 2008) Alvarez, Alejandra R.; Klein, Andres; Castro, Juan; Cancino, Gonzalo I.; Amigo, Julio; Mosqueira, Matias; Vargas, Lina M.; Yevenes, L. Fernanda; Bronfman, Francisca C.; Zanlungo, Silvana
    Niemann-Pick type C (NPC) disease is a fatal autosomal recessive disorder characterized by the accumulation of free cholesterol and glycosphingo-lipids in the endosomal-lysosomal system. Patients with NPC disease have markedly progressive neuronal loss, mainly of cerebellar Purkinje neurons. There is strong evidence indicating that cholesterol accumulation and trafficking defects activate apoptosis in NPC brains. The purpose of this study was to analyze the relevance of apoptosis and particularly the proapoptotic c-Abl/p73 system in cerebellar neuron degeneration in NPC disease. We used the NPC1 mouse model to evaluate c-Abl/p73 expression and activation in the cerebellum and the effect of therapy with the c-Abl-specific inhibitor imatinib. The proapoptotic c-Abl/p73 system and the p73 target genes are expressed in the cerebellums of NPC mice. Furthermore, inhibition of c-Abl with imatinib preserved Purkinje neurons and reduced general cell apoptosis in the cerebellum, improved neurological symptoms, and increased the survival of NPC mice. Moreover, this prosurvival effect correlated with reduced mRNA levels of p73 proapoptotic target genes. Our results suggest that the c-Abl/p73 pathway is involved in NPC neurodegeneration and show that treatment with c-Abl inhibitors is useful in delaying progressive neurodegeneration, supporting the use of imatinib for clinical treatment of patients with NPC disease.
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    Multiple Labeling of Compartmentalized Cortical Neurons in Microfluidic Chambers
    (2024) Moya-Alvarado, Guillermo; Aguirre-Soto, Alejandro; Bronfman, Francisca C.
    Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells.
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    p75 Neurotrophin Receptor-mediated Apoptosis in Sympathetic Neurons Involves a Biphasic Activation of JNK and Up-regulation of Tumor Necrosis Factor-α-converting Enzyme/ADAM17
    (2010) Kenchappa, Rajappa S.; Tep, Chhavy; Korade, Zeljka; Urra, Soledad; Bronfman, Francisca C.; Yoon, Sung Ok; Carter, Bruce D.
    During the development of the sympathetic nervous system, the p75 neurotrophin receptor (p75NTR) has a dual function: promoting survival together with TrkA in response to NGF, but inducing cell death upon binding pro or mature brain-derived neurotrophic factor (BDNF). Apoptotic signaling through p75NTR requires activation of the stress kinase, JNK. However, the receptor also undergoes regulated proteolysis, first by a metalloprotease, and then by gamma-secretase, in response to pro-apoptotic ligands and this is necessary for receptor mediated neuronal death (Kenchappa, R. S., Zampieri, N., Chao, M. V., Barker, P. A., Teng, H. K., Hempstead, B. L., and Carter, B. D. (2006) Neuron 50, 219-232). Hence, the relationship between JNK activation and receptor proteolysis remains to be defined. Here, we report that JNK3 activation is necessary for p75NTR cleavage; however, following release of the intracellular domain, there is a secondary activation of JNK3 that is cleavage dependent. Receptor proteolysis and apoptosis were prevented in sympathetic neurons from jnk3(-/-) mice, while activation of JNK by ectopic expression of MEKK1 induced p75NTR cleavage and cell death. Proteolysis of the receptor was not detected until 6 h after BDNF treatment, suggesting that JNK3 promotes cleavage through a transcriptional mechanism. In support of this hypothesis, BDNF up-regulated tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM17 mRNA and protein in wildtype, but not jnk3(-/-) sympathetic neurons. Down-regulation of TACE by RNA interference blocked BDNF-induced p75NTR cleavage and apoptosis, indicating that this metalloprotease is responsible for the initial processing of the receptor. Together, these results demonstrate that p75NTR-mediated activation of JNK3 is required for up-regulation of TACE, which promotes receptor proteolysis, leading to prolonged activation of JNK3 and subsequent apoptosis in sympathetic neurons.
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    The p75 neurotrophin receptor evades the endolysosomal route in neuronal cells, favouring multivesicular bodies specialised for exosomal release
    (2014) Escudero, Claudia A.; Lazo, Oscal M.; Galleguillos, Carolina; Parraguez, Jose I.; Lopez-Verrilli, Maria A.; Cabeza, Carolina; Leon, Luisa; Saeed, Uzma; Retamal, Claudio; Gonzalez, Alfonso; Marzolo, Maria-Paz; Carter, Bruce D.; Court, Felipe A.; Bronfman, Francisca C.
    The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and post-endocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.
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    The Rab11-regulated endocytic pathway and BDNF/TrkB signaling: Roles in plasticity changes and neurodegenerative diseases
    (2022) Moya-Alvarado, Guillermo; Guerra, Miguel V.; Tiburcio, Reynaldo; Bravo, Evelyn; Bronfman, Francisca C.
    Neurons are highly polarized cells that rely on the intracellular transport of organelles. This process is regulated by molecular motors such as dynein and kinesins and the Rab family of monomeric GTPases that together help move cargo along microtubules in dendrites, somas, and axons. Rab5-Rab11 GTPases regulate receptor trafficking along early-recycling endosomes, which is a process that determines the intracellular signaling output of different signaling pathways, including those triggered by BDNF binding to its tyrosine kinase receptor TrkB. BDNF is a well-recognized neurotrophic factor that regulates experience-dependent plasticity in different circuits in the brain. The internalization of the BDNF/TrkB complex results in signaling endosomes that allow local signaling in dendrites and presynaptic terminals, nuclear signaling in somas and dynein-mediated long-distance signaling from axons to cell bodies. In this review, we briefly discuss the organization of the endocytic pathway and how Rab11-recycling endosomes interact with other endomembrane systems. We further expand upon the roles of the Rab11-recycling pathway in neuronal plasticity. Then, we discuss the BDNF/TrkB signaling pathways and their functional relationships with the postendocytic trafficking of BDNF, including axonal transport, emphasizing the role of BDNF signaling endosomes, particularly Rab5-Rab11 endosomes, in neuronal plasticity. Finally, we discuss the evidence indicating that the dysfunction of the early-recycling pathway impairs BDNF signaling, contributing to several neurodegenerative diseases.
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    Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons
    (2021) Pinto, Cristina; Perez, Viviana; Mella, Jessica; Albistur, Miguel; Caprile, Teresa; Bronfman, Francisca C.; Henriquez, Juan Pablo
    The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.
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    TrkA receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal gamma-secretase-mediated release of the p75 intracellular domain
    (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2007) Urra, Soledad; Escudero, Claudia A.; Ramos, Patricio; Lisbona, Fernanda; Allende, Edgardo; Covarrubias, Paulina; Parraguez, Jose I.; Zampieri, Niccolo; Chao, Moses V.; Annaert, Wim; Bronfman, Francisca C.
    Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for gamma-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membrane-bound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where gamma-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for gamma-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.